The reproducible control of tumor blood flow, which is required for the optimal delivery of drugs and oxygen to solid tumors, remains an elusive goal. Nitric oxide (NO) is a multifunctional mediator of an array of physiological and pathological processes. It has been shown that NO derived from vascular endothelial NO synthase induces angiogenesis and maintains tumor blood flow. Recent results show that vascular NO mediates the recruitment of perivascular cells to angiogenic vessels, as well as the branching, longitudinal extension and subsequent stabilization of the vessels. However, preliminary studies suggest that NO produced by tumor and stromal cells outside the vascular area may compete with the process of vessel maturation. The central hypothesis of this project is that selective localization of NO around blood vessels improves tumor vascular morphology and function, the delivery of drugs and oxygen, and thus the efficacy of concomitant cytotoxic therapy. Orthotopic glioma and breast tumors grown in transparent window models in mice will be studied. In Aim 1, non-vascular sources of NO production will be blocked and vascular NO will be increased by means of genetic and pharmacological modifications. The effect of these modifications on tumor vascular function and tissue oxygenation will be determined by high-resolution intravital microscopy techniques and immunohistochemistry. Perivascular cells will be monitored in vivo using fluorescence-reporter transgenic mice. Finally, the tumor response to fractionated radiation during NO modifications will be determined. The angiopoietin (Ang)-Tie2 pathway has been shown to mediate vessel maturation via perivascular cell recruitment. In Aim 2, involvement of vascular NO in Ang-Tie2-induced perivascular cell recruitment as well as in downstream signaling of NO-dependent perivascular cell recruitment will be determined using recently established in vitro migration assays and a tissue-engineered blood vessel model as well as tumor models. Recent results show that blockade of vascular endothelial growth factor receptor 2 (VEGFR2) transiently improves perivascular cell coverage, tissue oxygenation and response to radiation therapy in tumors via Tie2 receptor signaling. In Aim 3, the role of vascular NO in anti-VEGFR2 treatment-induced vascular effects and the impact of vessel-selective localization of NO on the efficacy of combined anti-VEGFR2 and radiation treatments will be determined using the models and methods of Aim 1. In support of the central hypothesis, preliminary studies show that elimination of non-vascular sources of NO improves the structure and function of tumor vessels and enhances tumor response to fractionated radiation therapy. This project will advance the basic understanding of NO-mediated vessel maturation and help develop novel strategies to improve the delivery of drugs and oxygen to tumors.